The present invention relates to an insoluble matrix immobilized surfactant-coated lipase complex, to a method of preparing same and to the use of same as a biocatalyst for catalyzing, for example, inter- and/or trans-esterification of oils and fats in hydrophobic organic media. The novel procedures include two steps. In the first step, the enzyme is activated by being coated with a surfactant. In the second step, the enzyme is immobilized on the matrix of choice. These steps can be executed in any order.
Enzymatic modification of the structure and composition of oils and fats is of great industrial and clinical interest. This process is accomplished by exploiting regio-specific lipases in inter-esterification and/or trans-esterification reactions utilizing fats or oils as substrates (Macrea, A. R., 1983, J. Am. Oil Chem. Soc. 60: 291-294).
Using an enzymatic process, it is possible to incorporate a desired fatty acyl group on a specific position of a triacylglycerol molecule, whereas conventional chemical inter-esterification does not possess regio-specificity. Conventionally, chemical reactions are promoted by sodium metal, sodium alkoxide or cobalt chloride that catalyze acyl migration among triglyceride molecules, leading to the production of triglycenrdes possessing randomly distributed fatty acyl residues (Erdem-Senatalar, A., Erencek, E. and Erciyes, A. T., 1995, J. Am. Oil Chem. Soc. 72: 891-894).
In recent years, a number of studies have demonstrated the potential application of lipases as promising biocatalysts for different esterification reactions in organic media (Wisdom, R. A., Dunhill, P., and Lilly, M. D., 1987, Biotechnol. Bioeng. 29: 1081-1085).
Lipases with 1,3-positional specificity principally catalyze hydrolysis of fats and oils, to yield free fatty acids and glycerol. However, recent studies have shown that lipases with 1,3-positional specificity are also capable of catalyzing two types of esterification reaction in microaqueous organic media (Quinlan, P. and Moore, S., 1993, INFORM 4: 580-585). The first of these reactions is an inter-esterification or acidolysis reaction in which free fatty acids react with different triglycerides to yield new triglyceride molecules. The second type of reaction is trans-esterification in which two different triglyceride molecules react to give new triglyceride molecules (see FIGS. 1a-b). In both of these enzymatic reactions, the sn-2 position of the reacting triglycerides remains unchanged.
In general, water concentration plays an important role in determining the activity of enzymes. It also affects the equilibrium state of the reactions performed in hydrophobic organic solvents (Valiverty, R. H., Halling, P. J. and Macrae, A. R., 1993, Biotechnol. Lett. 15: 1133-1138). Since water is vital for the activity of enzymes in both hydrolysis as well as in synthesis reactions, as a compromise between hydrolysis and synthesis of triglycerides, the concentration of water is lowered so that the occurrence of undesirable reactions is minimized, but the water available is sufficient for the enzyme to remain active.
At high concentrations of water, e.g., above 5% of solvent weight, lipases possess preferably their natural hydrolytic activity, therefore, hydrolysis reaction proceeds. However, at low concentrations of water, e.g., below 1% of solvent weight, lipases catalyze the reverse reaction, that is, synthesis.
A typical range of water concentrations needed for promotion of inter-esterification reaction between different oils in organic media is 1-10 weight percent (wt %) of the hydrophobic organic solvent. This water concentration can normally facilitate also the hydrolysis reaction thus producing undesirable partial glycerides (mono- and di-glycerides) in the range of 10-20 wt % of the initial triglycerides concentration, as by products
The scope for exploiting the positional specificity of lipases, especially, in the food and oleochemical industries for the production of high-valued special fats is enormous. For example, cocoa butter substitute, simulated human milk fat and other structured triglyceride of specific nutritional quality can be obtained enzymatically by employing lipases with 1,3-positional specificity (Vulfson, E. N., 1998, Trends Food Sci. Technol 4.209-215)
In view of the foregoing, it is recognized that there is a need to develop new structured triglycerides with both medium-chain and -3 polyunsaturated fatty acids that would be devoid of the adverse effects of the naturally occurring -3 polyunsaturated fatty acids, or saturated fatty acids. For example, molecules of MCTs having one of their acyl groups substituted with an essential long-chain fatty acid would provide the nutritional advantages of both MCTs and LCTs. This approach is illustrated by the very useful triglyceride that is formed by incorporating the acyl form of the polyunsaturated fatty acids, EPA, DHA or -linolenic acid at the Sn-2 position of a triglyceride molecule having a medium-chain fatty acyl group at the sn-1 and sn-3 positions (Odle, J., 1997, J. Nut. 127: 1061).
The aforementioned polyunsaturated fatty acids incorporated into triglyceride molecules were shown to have several health benefits with respect to cardiovascular disease, immune disorders and inflammation, allergies, diabetes, kidney diseases, depression, brain development and cancer. Furthermore, medium-chain fatty acids incorporated into the same triglyceride molecule are of major importance in some clinical uses, especially, for facilitating absorbability and solubilization of cholesterol in blood serum, and for providing readily available energy sources for body consumption.
Many different approaches for the use of lipases in organic media have been attempted in order to activate them and to improve their performance.
These include the use of lipase powder suspended in either microaqueous organic solvents or in biphasic systems, and native lipases is adsorbed on microporous matrices in fixed- and fluidized-bed reactors (Malcata, et al., 1990, J. Am. Oil Chem. Soc. 890-910). Furthermore, lipases have been hosted in reverse micelles, and in some studies lipases were attached to polyethylene glycol or hydrophobic residues to increase their solubility and dispersibility in organic solvents.
None of the abovementioned approaches was found to be applicable for all enzymatic systems. However, in many cases, when lipases were treated in one way or another as described, their performance with respect to activity, specificity, stability and dispersibility in hydrophobic organic systems was improved.
In recent studies, the development of surfactant-coated lipase preparations has been reported (e.g., Basheer, S., Mogi, K. and Nakajima, M., 1995, Biotechnol. Bioeng. 45: 187-195). This enzyme modification converts slightly active or completely inactive lipases, with respect to esterification of triglycerides and fatty acids in organic media, into highly active biocatalysts. The newly developed surfactant-lipase complexes have been further studied and used for the inter-esterification reaction in organic solvent systems to produce structured triglycerides of major importance in medical applications (Tanaka, Y., Hirano, J. and Funada, T., 1994, J. Am. Oil Chem. Soc. 71: 331-834).
In another approach to the problem, various immobilized-enzyme reactor systems were used in lipase-catalyzed reactions in microaqueous hydrophobic organic media (e.g., Basheer, S., Mogi, K., Nakajima, M., 1995, Process. Biochemistry 30: 531-536). These included fixed- and fluidized-bed reactors, and a slurry reactor. In the published studies, lipase immobilized onto an inorganic matrix was used both in a batch reactor system, and in fixed-bed bioreactor systems. However, the lipases employed were not surfactant-coated and therefore have the same limitations as free lipase systems. These limitations include:
1. Difficulties in recovering the enzyme after completion of the process;
2. Rapid loss of activity of the free enzyme in the reaction medium;
3. Problems of recoverabilty of expensive enzymes;
4. Low synthetic activity of free lipases in organic solvents.
Neither of the abovementioned strategies has satisfactorily solved the technical problems encountered in directing trans- and inter-esterification of fats and oils. It is therefore an object of the invention to provide a lipase preparation that is capable of catalyzing esterification reactions in fats and oils with a much greater efficacy than existing methods.
It is another purpose of the invention to provide a lipase preparation that incorporates both immobilization to a matrix, and treatment by coating with a surfactant.
It is a further object of the invention to provide such a lipase preparation that may be used repeatedly, on an industrial scale with minimal loss of activity.
It is a further object of the invention to provide a method for preparing said insoluble matrix-immobilized, surfactant-coated lipase complex.
Yet a further purpose of the invention is to provide a process for preparing structured triacylglycerols, using said insoluble matrix-immobilized, surfactant-coated lipase complexes.
Other objects and advantages of the invention will become apparent as the description proceeds.
It has now been surprisingly found, and this is an object of the invention, that the dual modification of crude lipase by (1) coating with a surfactant, and (2) immobilization to an insoluble matrix; results in a synergistic improvement in the efficiency of the enzyme to catalyze trans- and inter-esterification reactions, when compared to either of these two treatments alone. It has been further unexpectedly found that it is possible to enhance the catalytic stability of said dually modified lipase for esterification reactions, by providing the enzyme preparation in a granulated form.
The invention is primarily directed to a lipase preparation comprising an insoluble matrix and a surfactant-coated lipase complex immobilized onto said insoluble matrix.
The immobilization of the lipase complex onto the insoluble matrix may be achieved by several different methods. According to a preferred embodiment of the invention, however, the surfactant-coated lipase complex is covalently, ionically or physically bound to the insoluble matrix.
The invention encompasses the use of many types of matrix, said matrices being selected from the group consisting of an inorganic insoluble matrix and an organic insoluble matrix.
In a preferred embodiment of the invention, the inorganic insoluble matrix is selected from the group consisting of alumina, diatomaceous earth, Celite, calcium carbonate, calcium sulfate, ion-exchange resin, silica gel and charcoal.
The abovementioned ion-exchange resin may be of any suitable material, but in a preferred embodiment is selected from the group consisting of Amberlite and Dowex.
Although any suitable organic insoluble matrix may be use, in a preferred embodiment of the invention, the organic insoluble matrix is selected from the group consisting of Eupergit, ethylsulfoxycellulose and aluminium stearate.
In a preferred embodiment, the content of the lipase is 2-20 weight percent of the surfactant-coated lipase complex. In a still more preferred embodiment, the content of the lipase is 0.01-1.0 weight percent of the preparation.
The invention provides the above-described lipase preparation, wherein the surfactant in the surfactant-coated lipase complex includes a fatty acid conjugated to a hydrophilic moiety. In a preferred embodiment, the fatty acid is selected from the group consisting of monolaurate, monomyristate, monopalmitate, monostearate, dilaurate, dimyristate, dipalmitate, distearate, trilaurate, trimyristate, tripalmitate and tristearate. In a preferred embodiment, the hydrophilic moiety is selected from the group consisting of a sugar, a phosphate group, a carboxylic group and a hydroxylated organic residue. In a more preferred embodiment, the sugar is selected from the group consisting of sorbitol, sucrose, glucose and lactose. Although the fatty acid and the hydrophilic moiety may be linked by any suitable type of bond, in a preferred embodiment, the fatty acid and the hydrophilic moiety are conjugated via an ester bond.
Although the lipase may be derived or obtained from any convenient source, in a preferred embodiment, the lipase is derived from a microorganism. Many different species of both microorganisms and multicellular organisms may be used as a source of lipase for the lipase preparation of the invention. The invention, however, is particularly directed to the use of lipase that is derived from a species selected from the group consisting of Burkholderia sp., Candida antractica B, Candida rugosa, Pseudomnonas sp., Candida antractica A, Porcine pancreas lipase, Humicola sp., Mucor miehei, Rhizopus javan., Pseudomonas fluor., Candida cylindrcae, Aspergillus niger, Rhizopus oryzae, Mucor jaanicus, Rhizopus sp., Rhizopus japonicus and Candida antractica. 
In a further aspect, the invention is directed to a lipase preparation comprising an insoluble matrix and a surfactant-coated lipase complex immobilized onto said insoluble matrix, said lipase preparation being provided in an organic solvent. In a preferred embodiment, the organic solvent is selected from the group consisting of n-hexane, toluene, iso-octane, n-octane, benzene, cyclohexane and di-iso-propylether. The invention is further directed to the use of said lipase preparation as a catalyst for esterification, inter-esterification and trans-esterification of oils and fats and alcoholysis of triglycerols and fatty alcohols. In a preferred embodiment, the lipase preparation is used as a catalyst with 1,3-positional specificity with respect to triacylglycerols.
In another aspect, the invention is directed to a lipase preparation as described above, wherein said preparation is in granulated form.
The invention also provides a lipase preparation, as described hereinabove, wherein the insoluble matrix has been modified with a fatty acid derivative.
In a further aspect the invention is directed to an enzyme preparation, as described hereinabove, for use in a reaction environment without the need for water addition.
The invention also encompasses a method for improving the stability of a surfactant-coated immobilized lipase complex, comprising granulating same prior to contacting it with the substrate to be reacted.
In a further aspect, the invention provides a method of preparing an insoluble matrix-immobilized surfactant-coated lipase complex comprising, in any desired order, the steps of:
(a) contacting a lipase in an aqueous medium with a surfactant, at a concentration and temperature, and for a period of time sufficient to obtain a coating of said lipase; and
(b) contacting said lipase in an aqueous medium, with an insoluble matrix, at a concentration, under conditions and for a period of time sufficient to obtain immobilization of said lipase on said matrix.
In a preferred embodiment of the abovementioned method, the lipase is first contacted with the insoluble matrix, and thereafter with the surfactant. In another preferred embodiment thereof, the lipase is first contacted with the surfactant, and thereafter with the insoluble matrix.
In a preferred embodiment of the invention, the above-described method further comprises the separation of the matrix-immobilized surfactant-coated lipase complex from the aqueous solution in which it was formed. In a still more preferred embodiment, this method also further comprises the step of drying said matrix-immobilized surfactant-coated lipase complex. Although the drying step may be accomplished by any convenient method, in a preferred embodiment, said drying is effected by freeze drying. In another preferred embodiment, the matrix-immobilized surfactant-coated lipase complex is dried to a water content of less than 100 parts per million by weight.
In another preferred embodiment, the aqueous solution used in the above-described method is a buffered-aqueous solution.
In yet another preferred embodiment of the above-described method, the lipase and surfactant are contacted in the aqueous medium by:
(i) dissolving said surfactant in an organic solvent for obtaining a dissolved surfactant solution; and
(ii) mixing said lipase and said dissolved surfactant solution in said aqueous medium.
In another preferred embodiment, the method further comprises sonication of the aqueous solution.
In yet another preferred embodiment of the method of the invention, the insoluble matrix is selected from the group consisting of alumina, diatomaceous earth, Celite, calcium carbonate, calcium sulfate, ion-exchange resin, silica gel, charcoal, Eupergit, ethylsulfoxycellulose, aluminium stearate and fatty acid derivative-treated Celite or other inorganic matrices.
In another preferred embodiment, the surfactant of the method includes a fatty acid conjugated to a hydrophilic moiety. In a still more preferred embodiment, said fatty acid is selected from the group consisting of monolaurate, monomyristate, monopalmitate, monostearate, dilaurate, dimyristate, dipalmitate, distearate, trilaurate, trimyristate, tripalmitate and tristearate.
In another preferred embodiment of the method of the invention, the hydrophilic moiety is selected from the group consisting of a sugar and a phosphate group and a carboxylic group and a polyhydroxylated organic residue. In a still more preferred embodiment, the sugar is selected from the group consisting of sorbitol, sucrose, glucose and lactose.
In another preferred embodiment of the method of the invention, the fatty acid and the hydrophilic moiety are conjugated via an eater bond.
In a preferred embodiment of the method of the invention, the lipase is derived from an organism. In a more preferred embodiment, the lipase is derived from a multicellular microorganism. Although the lipase may be derived from any suitable host, in a preferred embodiment, the lipase is derived from a species selected from the group consisting of Burkholderia sp., Candida antarctica B, Candida rugosa, Pseudomnonas sp., Candida antractica A, Porcine pancreatic lipase, Humicola sp., Mucor miehei, Rhizopus javan., Pseudomnonas flour., Candida cylindrcae, Aspergillus niger, Rhizopus oryzae, Mucor jauanicus, Rhizopus sp., Rhizopus japonicus and Candida antarctica. 
In another aspect, the invention is directed to a process for preparing structured triacylglycerols by esterification, acidolysis, trans-esterification, inter-esterification or alcoholysis between two substrates comprising contacting an insoluble matrix-immobilized surfactant-coated lipase complex with said substrates.
In a preferred embodiment of this process, the matrix-immobilized surfactant-coated lipase complex is contacted with the substrates in the presence of an organic solvent.
In another preferred embodiment of this process, at least one of the substrates is selected from the group consisting of an oil, a fatty acid, a triacylglycerol and a fatty alcohol. Although many different types of oil may be used in this process, in a preferred embodiment, the oil is selected from the group consisting of olive oil, soybean oil, peanut oil, fish oil, palm oil, cotton seeds oil, sunflower oil, Nigella sativa oil, canola oil and corn oil.
In another preferred embodiment, the fatty acid is selected from the group consisting of medium and short-chain fatty acids and their ester derivatives. In a still more preferred embodiment, the fatty acid is selected from the group consisting of oleic acid, palmitic acid, linolic acid, linolenic acid, stearic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid and their ester derivatives.
While the above-described process may be performed in any suitable receptacle, said process, in a preferred embodiment, is carried out in a tank reactor or in a fixed-bed reactor.
The invention also encompasses a triacylglycerol prepared according to the above-described process for use as a cocoa butter substitute, human milk fat-like triglycerides for special diets, or structured triglycerides for medical applications.